Immunogenic compositions of human papillomavirus and sera produced therefrom

ABSTRACT

A purified human papillomavirus gene selected from the group consisting of E1, E6-E7, Li, and L2, wherein said human papillomavirus is selected from the group consisting of HPV-2d, HPV-10b, HPV-14a, HPV-14b, HPV-15, HPV-17a, HPV-17b, HPV-19, HPV-20, HPV-21, HPV-22, HPV-23, HPV-24, HPV-28, HPV-29, HPV-31, HPV32, HPV-1P2 and HPV-IP4. Polypeptides encoded by these genes. The polypeptides can be used in immunogenic compositions.

The invention concerns papillomavirus DNAs and more particularly theprobes derived from these papillomaviruses, as well as the processeswhich use them for the in vitro diagnosis of papillomavirus infections.

The expression “papillomavirus” covers a great number of viruses havingin common being considered responsible for several forms of viralinfection ranging from relatively benign warts of the skin or mucousmembranes to hyperplasias susceptible to degenerating intointra-epithelial neoplasms or into various forms of skin cancer. To benoted also among the papillomavirus infections are particularly theepidermodysplasias verruciformis which will sometimes be referred tohereafter by the expression “EV”.

A certain number of types of papillomavirus have already been described.In the context of the present patent application, several new types andsub-types of papillomavirus will be described which have been isolatedfrom warts or disseminated macular lesions, likely to lead to thedevelopment of precocious skin cancers in a large proportion of affectedpatients.

Recent studies have revealed the importance of immune factors and themajor role of human papillomaviruses (HPV), to these factors added arethe role previously described in the literature of various geneticfactors and actinic radiations in the pathogenesis of papillomavirusinfections.

The invention is the result of observations regarding the relativebehavior of a great number of newly isolated papillomaviruses whoseessential genomic characteristics will be defined below.

The study of a small number of EV cases has already lead to thecharacterization of 6 types of HPV after molecular cloning of theirgenomes (KREMSDORF, D. et al. 1982, J. Virol. 43, 436-447, and KREMSDORFet al. 1983, J. Virol. 48, 340-351). These HPV have been divided intothree groups as a function of the lack of cross-hybridization or veryweak cross-hybridization between the genomes of the different groups.The first group includes the HPV3a and 10 which are associated with theplane warts observed in certain EV patients and in the generalpopulation; DNA sequences related to those of HPV3a have been found inthe cancer of an EV patient. The second group includes HPV5, 8 and 12,the genomes of HPV5 and 8 having been detected in the cancers of EVpatients. With the exception of a kidney transplant recipient presentingan immuno-suppression, who turned out to be infected by HPV5, the virusof the two latter groups have been detected only among EV patients, mostof them being infected by several viruses. It should be noted that amongthe 14 types of HPV currently mentioned in the literature (bibliographicreferences 1-5, 8, 9, 13, 14, 16, 18-20 indicated further on), four turnout to be specifically associated with EV which is a rare disease.

The labors which have led to this invention and which have permitted theisolation of a large number of new types and sub-types ofpapillomavirus, create the possibility of more highly refined in vitrodiagnostic techniques. More particularly, the invention providesperfected techniques for papillomavirus identification, for example, ofthose obtained from lesions ox biopsy sections, and allows for moreprecise diagnoses which may also result in better prognoses with regardto the possible evolution of the lesions in question.

As a general rule, it should be noted that, despite being very differentfrom each other, the papillomaviruses have sizes of the order of7000-8000 base pairs. In addition, their genomes may neverthelesspresent certain degrees of homology. In what follows, reference will bemade to evaluations of the percentage of homology between the varioustypes and sub-types of papillomavirus, these homology percentages resultfrom hybridization assays performed under conditions referred to as‘non-stringent’ or ‘non-strict’, or under hybridization conditionscalled “stringent” ou “strict”.

Among the papillomaviruses may be distinguished several types ofpapillomavirus, these may be distinguished by their percentages ofhomology as measured under strict or stringent conditions.Papillomaviruses which, under these conditions, present homologypercentages of less than 50%, belong to different types. It may be notedin this regard, that the homology percentages between viruses ofdifferent types may even fall to zero under said strict or stringentconditions. Under these same conditions, viruses showing homologypercentages of more than 50% are considered as belonging to the sametype and form the different syb-types within this same type.

Hybridization assays under non-strict or non-stringent conditionsimplies the mutual placing into contact of DNAs derived from two viralisolates under the following conditions as described by HEILMAN, C. A.et al. 1980, J. Virol. 36, 395-407, and CROISSANT et al. 1982, C.R.Acad. Sc. Paris, 294, 81-586 (hetero-duplex molecules).

Hybridization assays under strict or stringent conditions imply theplacing into mutual contact of DNAs derived from two viral isolatesunder the conditions described by KREMSDORF, D. et al. (1982, J. Virol.43, 436-447, and J. Virol., 48, 340-351) and DAVIS, R. W. et al. 1971,Methods Enzymol. 21, 413-428 (hetero-duplex molecules).

Schematically, it may be noted that those papillomaviruses belonging toone same type presenting hybridizable sequences having virtuallyidentical nucleotide sequences over 80 to 100% of the totality of theirrespective lengths, these homologous sequences may be reduced to 60% orless among papillomaviruses of different types. The degree of identityor of analogy of the sequences from papillomaviruses of different typeswhich mutually hybridize under non-strict or non-stringent conditionsmust obviously be less than in the case of papillomaviruses of the sametype.

The study to which the inventors proceeded has shown both that thedegree of genetic heterogeneousness between diverse types ofpapillomaviruses was greater than previously recognized and at the sametime that the different types were fond often to be associated withforms or variants of infections presenting a certain degree ofspecificity.

The invention consequently concerns not only the DNAs susceptible tobeing isolated from different new papillomaviruses which have beenisolated and the probes which can be partially or entirely constitutedof these DNAs, but also mixtures or “cocktails” of papillomavirus typeslikely to be most effectively used for the diagnosis of diversecategories of infection, and of the levels of risk to the patient whichaccompanies the discovery of a given papillomavirus. The number ofpapillomavirus probes described in the present application, to which maybe added, as the case may be, those constituted from the genomic DNAs ofpapillomaviruses which have already been previously isolated, and theirassociations in the specific mixtures lend greater precision to thediagnosis, notably a greater discrimination between the diversecategories of infections which may be imputed to the diverse types ofpapillomavirus or which may be susceptible to develop under the effectof these latter types and, within a given category of infections to givea better prognosis of the degree of risk that these latter could betransformed to more serious disease. For example, the invention aims toprovide the means permitting, in the case of infections manifesting asepidermodysplasias verruciformis, to better appreciate the degree ofrisk that they may evolve towards skin cancer.

In a general manner and in an attempt to simplify the followingpresentation, the whole genomes of the papillomaviruses will bedesignated by the abbreviation “HPV-DNA”.

With the same aim of simplification, reference will be made to appendeddrawings on which figure physical restriction maps of HPV-DNAs includingsome from previously known papillomaviruses.

These physical maps give the positions of the fractionation sites of thevarious restriction endonucleases. The origin of the maps is generallyconstituted of the site of a single cut. The distances from this pointof origin are expressed as a percentage of the total length of thegenome. One unit of the map represents 1% of the length of the genome.

The invention first of all concerns most specifically each of theHPV-DNAs chosen from among the total of the DNAs having sizes rangingbetween 7000 and 8000 base pairs and are characterized by therestriction maps appearing in the drawings which concern moreparticularly HPV-DNAs obtained from papillomaviruses and whichcorrespond to the designations HPV-2d, HPV-10b, HPV-14a, HPV-14b,HPV-15, HPV-17a, HPV-17b, HPV-19, HPV-20, HPV-21, HPV-22, HPV-23,HPV-24, HPV-28, HPV-29, HPV-31, and HPV-32, HPV-IP2 and HPV-IP4.

It goes without saying that the invention equally extends its effects toHPV-DNAs which may be considered as belonging to the same types as thosejust enumerated.

The physical maps corresponding to the HPV-DNAs of newly characterizedviruses are indicated by a solid black circle on the drawings.

The invention equally concerns fragments of the preceding HPV-DNAs orcapable of hybridizing with them, notably under strict conditions.Likewise it concerns those recombinant DNAs containing all or part ofany of the HPV-DNAs indicated above, and more particularly recombinantDNAs containing fragments corresponding to genes E1, E6-E7, L1, and L2respectively or again fragments containing sequences corresponding tothe inter-gene regions of these said HPV-DNAs. Lastly it concerns theprobes which may be constituted from these respective HPV-DNAs, and theprocesses of in vitro diagnosis using said probes.

Preparations of viral DNA have been selectively extracted (LUTZNER, M.A., 1983, Lancet ii:422-424) from the products of scrapings of benignlesions of six European EV patients and two South American EV patients.The HPV-DNAs were purified by equilibrium centrifugation through cesiumchloride gradients and/or by sedimentation through sucrose gradients inthe presence of ethidium bromide, according to the operating procedurespreviously described (articles by KREMSDORF, D. et al. mentioned above,and ORTH, G. et al. 1980, Cold Spring Harbor Conf. on Cell Proliferation7:259-282). The DNA preparations were treated with restrictionendonucleases and the digestion products separated by electrophoresis onagarose gels (articles by KREMSDORF et al. already mentioned). Inaddition, from the HPVs 5, 8 and 12 (KREMSDORF et al. articles again)and HPV-2 (HEILMAN, C. A. et al. 1980, J. Virol. 36:395-407, and ORTH,G. et al. 1980, Cold Spring Harbor conf. on Cell Proliferation7:259-282) found in the verruca warts of one of the patients, elevenstrains different from those from previously characterized types, wereidentified furnishing the major models for the restriction enzymecleavage of the DNAs. The new HPV types were given a number and thesub-types of a type were given the same number followed by a letteraccording to the chronological order of their identification (COGGIN, J.R. et al. Cancer Res. 39:545-546). The genomes of the 11 new HPVs werecloned into Escherichia coli K12, strain C600 (KREMSDORF et al. article(1983) previously mentioned). The DNAs were inserted in the form ofmolecules of unitary length with the exception of two fragments of DNAfrom HPV-24 produced by endonuclease BamHI. They were inserted eitherinto plasmid pBR322 (SUTCLIFFE, J. G. 1978, Nucleic Acids Res.5:2721-2728), using the unique cleavage sites from AvaI, from BamHI, andfrom HindIII, or into a recombinant plasmid having integrated theHindIII B fragment from the DNA of HPV-5 (KREMSDORF et al. (1982)previously mentioned), which contains a unique SacI site. Moreparticularly, the HPVs 17b and 22 were inserted in the form of DNAmolecules of unitary length after splitting with an enzyme (SacI) whichsplits only once the DNA of HPVs and the recombinant plasmid pBR322containing the HindIII B fragment from the DNA of HPV-5. The DNA ofHPV-14a was inserted into the plasmid pBR322 in the form of a DNAmolecule of unitary length after incomplete digestion of the viral DNApreparation by HindIII, an enzyme which produces two fragments of 96.1and 3.9% of the length of the genome. the BamHI A and B fragments ofHPV-24 (having sizes corresponding to 83.1 and 16.9% respectively of thegenome length) were inserted separately into plasmid pBR322.

The isolated clones and the corresponding HPV sources are listed in theappended Table I. TABLE 1 ORIGIN OF DNAs FROM HPV CLONES Type of DNAfrom HPV Clon- Other types of Pa- ing^(c) HPV found in tient^(a)Nationality Source^(b) cloned enzyme the patients 1 Polish warts: knees14a Hind III 5 15 Bam HI 2 French warts: hands 14b Bam HI 3 Columbianmacules: trunk 17a Bam HI 5 4 Italian macules: breast 17b Sao I 5 22 SaoI 5 Dutch macules: back 19 Bam HI 5, 8, 17a macules: breast 24 Bam HI 6Columbian warts: hands 20 Ava I 5, 8, 24 7 Polish warts: knees 21 Bam HI2, 12, 17a, 20 8 Polish macules: 23 Bam HI 5, 8, 20 forearm

To identify the recombinant plasmids, the electrophoretic mobilities ofthe digestion products of the recombinant DNAs and the non-clonedHPV-DNAs were compared after treatment with a mixture of two restrictionendonucleases including the endonuclease used for the insertion of theviral sequences into the plasmid. The number and the sizes of theisolated fragments indicated that in each case the entire viral genomeswere integrated. A heterogeneity of the DNA sizes was observed whennon-cloned HPV-DNAs or those excised from plasmidic sequences, wereanalyzed by agarose gel electrophoresis (data not shown). The DNAs fromHPVs 14b, 19, 20 and 21 have sizes similar to those of HPVs 3a, 5, 8 and12 (around 7700 nucleotide pairs) (articles by KREMSDORF (1982) and ORTH(1980) already mentioned).

The sensitivity of cloned viral genomes to 14 restriction endonucleaseswas analyzed and physical maps established (FIGS. 1 through 10). Therestriction maps of certain HPV-DNAs are repeated in some of the figuresfor reasons which will be explained further on. Between 22 and 33cleavage sites have been localized according to the methods previouslydescribed (9). No apparent analogy could be detected between the mapswith the exception of those of HPVs 14a and 14b, on the one hand (FIGS.4 a and 4 b), and those of HPVs 17a and 17b, on the other (FIG. 5).Among the 21 and 31 sites localized respectively on the DNAs of the HPVs14a and 14b, fifteen turned out to be in common when one of the twoBamHI cleavage sites of the DNA of HPV-14a was aligned with the uniqueBamHI cleavage site of the DNA of HPV-14b. In a similar manner, 21 ofthe 29 cleavage sites on the DNA of HPV-17a were equally found on theDNA of HPV-17b (out of 26 sites), when the sites of the unique BamHIcleavages wer aligned.

No apparent analogy has been detected between these maps and thosepreviously established for the HPVs associated with EV (HPVs 3a, 5, 8,9, 10 and 12) (8, 9, 16, 18 and 20), with warts on the skin (HPVs 1, 2,4 and 7), with lesions of the muco-cutaneous or mucous membranes (HPVs6b, 11a, 13 and 16), with the exception of the map for HPV-14a which isclosely related to the map of an HPV isolated from a Japanese male withEV (24). This latter isolate differs from HPV-14a by one additionalBamHI site and one additional HindIII site, while the locations of thesites: AvaI, BamHI, BglI, EcoRI, HindIII and HindIII were similar forthe two viruses. Cross-hybridization experiments confirmed that thesetwo viruses are indeed closely related.

It should be noted that some sites (those indicated by arrows) were notlocated. Cleavage sites differing be less than 2% of the length of thegenome were considered as retained (*). Enzymes which produced nosplitting were: PvuI, SalI, and SmaI for the DNA of HPVs 14a and 23;PvuI, SacI, SalI and SmaI for the DNA of HPV-14b; BglI, PvuI, SalI andSmaI for the DNA of HPVs 15, 17a and 17b; BglI, SacI, SalI and SmaI forthe DNA of HPV-19; EcoRI, PvuI, SacI and SmaI for the DNA of HPV-20;SacI and SmaI for the DNA of HPV-21; BamHI, BglI, PvuI, PvuII and SalIfor the DNA of HPV-22; BglI, EcoRI, PvuI, SacI and SmaI for the DNA ofHPV-24.

The existence of sequence homologies between the DNAs of the newlycharacterized HPV-DNAs as well as between these and the previouslycharacterized HPV-DNAs of EV (HPVs 3a, 5, 8, 9, 10 and 12), of HPVsassociated with skin warts (HPVs 1, 2, 4 and 7), and of HPVs associatedwith mucous membrane lesions (HPVs 6b, 11a, 13 and 16) has been studied.Hybridization experiments by attachment onto filter paper and DNA-DNAhybridization in saturated liquid phase followed by S1 nucleasedigestion were carried out under the strict or stringent conditionspreviously described (8, 9). In particular, the DNAs of HPVs werelabeled by “nick translation” and separated by sedimentation on alkalinesucrose gradients (5 to 20%) as previously described (13). The labeled(4000 cpm) HPV-DNAs were incubated in NaCl 0.48M/EDTA 1 mM (pH 6.8) at68 degrees C., in the presence of either calf thymus DNA (20micrograms), or unlabeled HPV-DNA (0.20 micrograms) as previouslydescribed (8, 9). The specific activity of the HPV-DNA probes variedbetween 5.3×10⁷ and 2×10⁸ cpm/microgram. The percentage of hybridizationwas determined by measure of the fractions resisting the S1 nuclease.The numbers represent the values corrected for spontaneous renaturation(4 to 15%) of the probes and normalized to 100% for the homologoushybridization (75 to 95%). The abbreviation ND signifies “notdetermined”. The relative amounts of cross-hybridization between theHPV-DNAs under the conditions indicated above are expressed as a % ofhybridization between the labeled HPV-DNA and the unlabeled HPV-DNA.TABLE 2 DEGREE OF CROSS-HYBRIDIZATION BETWEEN DNAs FROM HPV, DETERMINEDBY LIQUID PHASE HYBRIDIZATION. Unlabeled % of hybridization with DNAfrom HPV labeled with ³²p HPV DNA 3a 5 14a 14b 19 20 21 22 23 9 15 17a17b 24  1a 0.1 0.3 0.2 0.3 0 0.8 2.9 0 0 1.0 0.4 0.2 0 0 11a 1.6 1.0 1.30 0.9 0.1 0.7 3.7 0 0.1 0.1 0.6 3.3 0  3a 100 1.8 1.0 0 1.5 0.1 1.5 01.9 0.1 1.7 1.2 1.8 3.0 10 32.3 ND ND 0.1 0 0.1 1.9 3.0 ND 0 1.6 0.1 2.0ND  5 0.2 100 12.1 12.4 5.8 9.3 9.4 10.1 5.0 4.3 0.7 8.6 0 2.4  8 1.115.7 9.9 13.4 8.5 5.6 5.0 7.1 5.8 3.5 1.5 3.2 3.8 0.1 12 0.1 19.3 9.212.5 5.3 8.6 9.3 11.7 4.0 3.6 1.2 0.1 1.9 0 14a 0.2 13.2 100 88.0 14.682.4 32.9 10.1 24.6 8.0 2.2 2.4 4.1 ND 14b ND 10.5 94.1 100 9.8 28.435.4 9.5 28.2 0 0 0 0 0 19 ND 7.2 21.4 20.6 100 7.6 8.8 15.5 27.7 0 0 02.2 1.0 20 ND 9.9 28.8 37.9 6.2 100 25.4 13.7 14.1 0 0 2.1 0 3.6 21 ND10.5 38.7 40.5 6.4 37.5 100 9.8 18.6 0.1 0 2.5 0.3 0 22 ND 7.2 7.4 ND17.3 7.2 10.0 100 17.9 0 0 0.1 0.1 0 23 ND ND ND ND ND ND ND 21.2 100 00.5 0 0 0.1  9 0.4 3.1 0.5 1.2 0 2.0 1.0 0 0 100 5.5 6.3 5.4 0 15 0.43.3 2.1 3.3 0 0.1 0.8 0 0 7.8 100 22.5 21.6 0 17a 0.7 1.2 1.4 2.8 0 0.10.1 0.8 1.4 7.6 19.5 100 92.7 0 17b ND ND ND 1.4 0 0.3 3.4 ND ND ND ND86.3 100 ND 24 ND ND 0.1 2.6 ND ND ND 0.8 0 0.2 0 1.1 1.1 100ND = not determined.

The absence or near absence of cross-hybridization between the genomesof HPVs 1, 2, 4, 6b, 7 and 11, and the HPV-DNAs of 32 P labeled newlycloned EV, or between unlabeled HPV-DNAs of EV and probes specific toHPVs 13, 16 and 18 is to be noted. In a similar manner, almost nocross-hybridization has been detected between the DNAs of HPVs 14a, 14b,15, 17a, 17b, 19, 20, 21, 22, 23 and 24, and the DNAs of HPVs 1a and 11aby saturation reassociation (Table 2). The DNAs of newly cloned HPVsshowed little or no cross-hybridization or showed less than 50%cross-hybridization between each other and with the genomes of otherHPVs associated with EV (HPVs 3a, 5, 8, 9, 10 and 12) with the exceptionof HPVs 14a and 14b on the one hand, and HPVs 17a and 17b on the other,which showed strong cross-hybridization. These observations justify theclassification of the new viruses into nine new types (HPVs 14, 15, 17,19, 20, 21, 22, 23 and 24) plus two sub-types of types 14 (HPV-14a and14b) and 17 (HPV-17a and 17b).

Likewise, the different HPVs have been classed into groups based ontheir homologies (or lack of them) of sequences under the strictconditions of molecular hybridization. These groups, designated by theletters A to H, are listed in Table 3 which is appended. This Tablementions the diseases which have been diagnosed among the carriers ofthese HPVs (separately or in combination) and their oncogenic potential.TABLE 3 CLASSIFICATION OF HPVs WHICH ARE THE OBJECT OF THIS PATENTAPPLICATION, AS A FUNCTION OF THE DEGREE OF HOMOLOGY OF THEIR NUCLEOTIDESEQUENCES AS DETERMINED BY MOLECULAR HYBRIDIZATION UNDER STRICTCONDITIONS Homologies within the Probe Group¹⁾ HPV types²⁾ groupAssociated diseases Oncogenic potential mixture A  1 Myrcemies very weak1 B  2 Verruca warts weak 1 C  3, 10, 28*, 29* 14 to 38% Plane wartsmoderate; 2 Intermediary warts a related virus, associated Actinickératoses with intra-epithelial Bowen's disease neoplasms and evolvingskin cancers D  4 Verruca warts very weak 1 E  5, 8, 12, 14*,  4 to 18%Epidermodysplasia verruciformis HPVs 5, 8 and 14 associated 3, 4, 7 19*,20*, 21*, Actinic kératoses with cancers from E.V.; an 22*, 23* Bowen'sdisease related virus, associated Skin cancers with intra-epithelialneoplasmas and evolving skin cancers F  9, 15*, 17*,  6 to 23%Epidermodysplasia verruciformis 5 G 24* Epidermodysplasia verruciformis6 H 13, 31* Oral epithelial hyperplasia; Oral leucoplasias 8 I 32*Bowen's disease Intraepithelial neoplasm 7, 9 and skin cancers¹⁾Genomes of HPV types belonging to different groups, generally presentno detectable sequence homology under strict molecular hybridizationconditions. Genomes of HPV types belonging to the same group show lessthan 50% sequence homology.²⁾The new HPV types are indicated by an asterisk.

The DNAs of HPVs 5, 8, 12, 14, 19, 20, 21, 22 and 23 show between themlevels of cross-hybridization (group homologies) varying from 5 to 38%and show no notable cross-hybridization (4 to 13%) except with the DNAsof HPVs 5, 8 and 12. These viruses thus form a part of a group of HPVsof EV previously defined (9).

Likewise, the DNAs of HPVs 9, 15 and 17 which show between themcross-hybridization of around 20% and cross-hybridization of around 6%with the DNA of HPV-9, equally belong to a group of EV HPVs previouslydescribed (9). The HPVs of types 13 and 31 may be considered asbelonging to one same group. And last, the HPVs of types 1, 2, 4, 24 and32 which show almost no homology with the genomes of the other HPVs, areconsidered as forming the first members of other groups which aredistinct from each other and from the previous groups.

The invention concerns still more particularly the DNA fragments derivedfrom the above described HPV-DNAs, and especially those corresponding togenes E6-E7, E1, L2, L1 and to their inter-gene regions. The relativepositions and lengths of these various fragments in relation to thesites taken as origins (FIGS. 1 to 9) are indicated in Table 4 appended.TABLE 4 PUTATIVE LOCATION OF THE PRINCIPAL GENES AND OF THE INTERGENEREGIONS ON THE PHYSICAL MAPS OF THE HPV GENOMES. Co-ordinates of the 5′and 3′ ends, corresponding to genes Type of HPV E6-E7 E1 L2 L1 Intergeneregion  1   44-34.5 35-11   95-75.5 76.5-56   56-44.5  3 18.5-9  9.5-85.5 69.5-50 51-30.5 30.5-19    5  6.5-97 97.5-73.5 57.5-30 39-18.518.5-7    8   63-53.5 54-30   14-94.5 95.5-75   75-63.5  9   42-32.533-9    93-73.5 74.5-54   54-42.5  10a   49-39.5 40-16   0-80.581.5-61   61-49.5  10b   93-83.5 84-60   44-24.5 25.5-5    5-93.5 1223.5-14 14.5-90.5 74.5-55 56-35.5 35.5-24   14  8.5-99 99.5-75.5 59.5-4041-20.5 20.5-9   15 39.5-30 30.5-6.5  90.5-71 72-51.5 51.5-40   17  46-36.5 37-13   97-77.5 78.5-58   58-46.5 24 24.5-15 15.5-91.5 75.5-5657-36.5 36.5-25   28 47.5-38 38.5-14.5 98.5-79 80-59.5 59.5-48   2989.5-80 80.5-56.5 40.5-21 22-1.5  1.5-90  31   89-78.5   80-53.5  33.5-15.5 17.5-96.5   96.5-92.5  

The localization of genes on the genome of HPV-1 has been deduced fromthe nucleotide sequence of this genome (patent of O. DANOS, M. KATINKAand M. YANIV). The physical maps of the genomes of the HPVs 3, 5, 8, 9,10a, 12, 14, 15, 17 and 24 have been aligned relative to the physicalmap and the gene map of HPV-1, and that of HPV-31 relative to thephysical map and gene map of HPV-6b (E. SCHWARZ and al, EMBO J., 1983,2, 2341-2248), after electron microscope analysis of the hetero-duplexmolecules formed under strict conditions (Tm −29 degrees C.) or lessstrict (Tm −40 degrees C.) of hybridization. The physical maps of HPVs10b, 28 and 29 were lined up against the pysical maps of HPVs 3a and 10aafter juxtaposition of the retained restriction enzyme sites.

The values of the co-ordinates shown in table 4 give the position, onthe physical amps presented in FIGS. 1-9, the 5′ and 3′ ends of thgenome segments homologous with the genes E6 and E7, E1, L2 and L1 andwith the inter-gene region relative to the genome of HPV-1a, or, in thecase of HPV-31, relative to the genome of HPV-6b.

The inter-gene region (which includes the elements of regulation) andthe adjacent E6 and E7 genes (corresponding probably to the major genesof transformation expressed in tumors) show no sequence homologydetectable by electron microscope analysis of the hetero-duplexmolecules formed (under non-strict hybridization conditions) between thegenomes of HPV types belonging to different groups, or formed (understrict hybridization conditions) between the genomes of most of the HPVtypes belonging to the same group. Gene E1 (implicated principally inthe replication of viral DNA) and gene L1 (coding for a major protein ofthe viral capsid bearing the virion's principal antigenic determinants)show sequence homologies detectable by the analysis of hetero-duplexesformed (under non-strict hybridization conditions) between the genomesof HPV types belonging to different groups, or (under strict conditions)between the genomes of HPVs belonging to the same group.

Probes prepared from recombinant plasmids which include the E1 and L1regions could theoretically permit the detection of the greatest numberof HPV types by molecular hybridization experiments under strict ornon-strict conditions according to the case. Probes prepated fromrecombinant plasmids which include the inter-gene region and the genesE6 and E7 permit the specific detection of one HPV type or related HPVtypes.

The L2 region (coding for a minor constituant of the viral capsid) showsa variable degree of conservation of nucleotide sequences among thedifferent HPV types.

In what follows, the conditions under which the viruses HPV-IP2 andHPV-IP4 have been isolated will be described more precisely, as will theconditions under which the HPV-DNAs from these viruses were obtained.

Molecular Cloning and Characterization of a New Type of HPV Associatedwith Neoplasms and Genital Cancers (HPV-IP2).

A new type of HPV has been demonstrated in the DNA extracted from acancer of the cervix, by hybridization under non-strict conditions witha radioactive probe specific to HPV type 16. No cross-hybridization wasdetectable when the hybridization was carried out under the strictconditions of hybridization. A study of the sensitivity of the DNA ofthis HPV to a number of restriction enzymes has shown that the enzymeBglII cut once the viral DNA. After digestion of the DNA extracted fromthe tumor, by the endonuclease BglII, the fraction containing the DNAmolecules of 8 kb (size of a genome of papillomavirus) were purified bycentrifugation through a sucrose gradient. The 8 kb molecules wereinserted, at the BglII site, into a vector constituted of the plasmidPL15.5 (which includes a single cut site by BglII and by BamHI) whichwas inserted by it's BamHI site into the DNA of the bacteriophage lambdaL47.1. After encapsidation of the recombinant DNA and infection of thehost bacteria Escherichia coli strain (LA101), the areas of lysiscorresponding to recombinant phages were detected by hybridization ofthe replicated infected bacterial cultures, with DNA from radioactiveHPV-16, under non-strict conditions. Several recombinant bacteriophages,containing the totality of the viral sequences, have been isolated: thecutting of the phagic DNA by the insertion enzyme BglII results in afragment of 8 kb hybridizing with the HPV-16 under non-strictconditions; the cutting of the DNA of the recombinant phages and of theDNA of the tumor of origin by a mixture of the enzymes BglII and PstIleads to the same 5 fragments, the total molecular weight of which isequal to the size of a genome of the papillomaviruses. The DNA of thenew HPV was excised from the DNA of the recombinant bacteriophages,purified by electro-elution, and re-cloned in the plasmid PL15.5. Arestriction map of the viral DNA has been established based on thesensitivity of this DNA to 18 restriction endonucleases, permitting 21cut sites to be localized (FIG. 9). The map thus established isdifferent from the map of the genomes of HPVs identified up til now. Thesequence homology between the DNA of the new HPV and the DNA of the HPVsidentified up til now were analyzed by molecular hybridizationexperiments on culture replicas under strict conditions. The homologydetected was always less than 5%, the greatest homology was detectedwith the genome of HPV-16. The new virus characterized from a cervicalcancer therefore constitutes a new type of HPV, provisionally calledHPV-IP2.

The analysis, by electron microscopy, of the hetero-duplex moleculesformed under different conditions between the DNA of HPV-IP2 and the DNAof HPV-1 permitted the alignment of the physical maps of these twogenomes and the definition of the theoretical positions of the differentgenes of the DNA of HPV-IP2.

Putative Positions of the Principal Genes and of the Inter-Gene Regionof the HPV-IP20N the Map of this Genome. Co-ordinates of the ends: 5′and 3′ E6-E7 62 71.5 E1 71 95 E2 95.5 11.5 L2 11 30.5 L1 31.5 52Inter-gene region 52 63.5

The use of radio-active probes prepated from the DNA of purified HPV-IP2has permitted the determination of the pathogenic power of theseviruses. The DNA of HPV-IP2 was found in one case of Bewenoid papula ofthe external genital organs, out of 14 cases studied; in two cases outof 51 of invasive cervical cancer studied; and in 1 case of cervicalintra-epithelial neoplasm out of 28 studied. Thus HPV-IP2 constitutes agenito-tropic type of HPV with oncogenic potential whose frequency is alittle less than that of HPV-18, and much less than that of HPV-16. Itis necessary to include it in any mixture of HPV-DNAs intended for thepreparation of molecular probes for the diagnosis of, or screening for,the types of HPVs presenting a risk of the development of genitalneoplasms and, in particular, of cervical cancer.

Molecular Cloning and Characterization of a New Type of HPV Associatedwith Pre-Cancerous Lesions of the Skin (HPV-IP4).

A new type of HPV has been found in the DNA extracted from the biopsy ofan actinic keratosis, a pre-cancerous skin lesion, by molecularhybridization under strict conditions, with a mixture containingradioactive probes specific for HPV types 5, 8 and 14. Nocross-hybridization was detected when the hybridization was carried outwith probes specific to types 1, 2, 3, 7, 10, 13, 16, 18, 28, IP-1(previously named HPV-31), IP-2 and IP-3 (previously named HPV-32).

A study of the sensitivity of this HPV to several restriction enzymeshas shown that the enzyme EcoRI cuts the viral DNA once. After digestionof the DNA extracted from the biopsy by the endonuclease EcoRI, thefraction which contains the molecules of DNA of 8 kb (size of a genomeof papillomavirus) was purified by centrifugatin through a sucrosegradient). The 8 kb molecules were inserted, by the EcoRI site, into theDNA of the bacteriophage lambda gt wes. lambda B. After encapsidation ofthe recombinant DNA and infection of the host bacteria (Escherichiacoli, strain LA101), the areas of lysis corresponding to the recombinantphages were detected by hybridization of the replicated infectedbacterial cultures, with a radio-active mixture of DNAs from the HPVs 8and 14 under non-strict conditions. Several recombinant bacteriophages,containing the totality of the viral sequences, have been isolated: thecutting of the phagic DNA by the insertion enzyme EcoRI results in an 8kb fragment hybridizing with the probe specific to HPVs 5, 8 and 14under non-strict conditions; the cutting of the DNA of the recombinantphages and the DNA of the original lesion by a mixture of the enzymesEcoRI and PstI results in the same 6 fragments, the total molecularweight of which equals the size of a genome of the papillomaviruses. TheDNA of the new HPV was excised from the DNA of a recombinantbacteriophage, purified by electro-elution, and re-cloned into theplasmid pSP65. A restriction map of the viral DNA was established fromthe sensitivity of this DNA to 15 restriction endonucleases, which haspermitted the localization of 23 cut sites (FIG. 10). The map thusestablished is different from the map of the genomes of HPVs identifiedup til now. The sequence homology between the DNA of the new HPV and theDNAs of the HPVs identified up to now has been analyzed by molecularhybridization experiments on cultures under strict conditions. Ahomology, less than 50%, has been detected between the DNA of the newHPV and the DNA of certain types of HPVs previously identified in thelesions of epidermoplasia verruciformis (HPVs 5, 8, 12, 14, 19, 20, 21and 25), but no homology was detected with other HPV types. The newvirus characterized from an actinic keratosis thus constitutes a newtype of HPV provisionally named HPV-IP4.

The use of a radioactive probe prepared from the DNA of purified HPV-IP4has permitted the demonstration of HPV-IP4 in 42% of the 17 patientsstudied having epidermodysplasia verruciformis and in x out of ybiopsies of actinic keratosis analyzed. Because of its great frequencyamong patients of EV, a disease characterized by the frequentdevelopment of skin cancers, and because of its association with afraction of the lesions of actinic keratosis considered as precursors ofspinocellular cancers of the skin. HPV-IP4 constitues a type ofdermo-tropic HPV with oncogenic potential. It is necessary toincorporate it into any mixture of HPV-DNAs intended for the preparationof molecular probes for the diagnosis or screening of HPV typesconstituting a risk of the development of cancerous or pre-cancerousskin lesions.

The invention still more particularly concerns the mixtures or cocktailsof different HPV-DNAs (or probes containing these HPV-DNAs or sequencesfrom them), susceptible to being used in combination to create means forthe global diagnosis of the different forms of papillomavirusinfections, possibly for use in the prognosis of the possible evolutionof the infection. The preferred mixtures conforming to the invention areidentified in Table 5 appended. TABLE 5 CHARACTERISTICS OF THE HPV-DNAMIXTURES USEABLE FOR THE DETECTION OF PAPILLOMAVIRUS INFECTIONSDesignation of the mixtures Constitution¹⁾ Diagnosable diseases 1  1,2d*, 4 Skin or mucous membrane warts (especially verruca and plantarwarts). Differential diagnosis of epidermodysplasia verruciformis. 2  3,10a, 10b*, Plane warts or intermediary skin or 28*, 29* mucous membranewarts. Intra-epithelial neoplasms and skin cancers. Differentialdiagnosis of epidermodysplasia verruciformis. 3  5, 17a*, 24*Epidermodysplasia verruciformis. Intra-epithelial neo-plasms and skincancers. 4  5, 8, 12, 14a*, Epidermodysplasia verruciformis. 14b*, 19*,20*, 21*, 22*, 23* 5  9, 15*, 17a*, 17b* Epidermodysplasiaverruciformis. 6 24* Epidermodysplasia verruciformis. 7  5, 8, 14b*, 32*Skin cancers from epidermodysplasia verruciformis. Intra-epithelialneoplasias and skin cancers. 8 13, 31* Oral epithelial hyperplasia;differential diagnosis of oral epithelial neoplasms. 9 32*Intra-epithelial neoplasms and skin cancers.¹⁾The new types of HPV are indicated by an asterisk.

This Table also indicates the natures of the affections susceptible tobeing more particularly diagnosed by the use of the mixtures figuring onthe left side of the table. It should be noted that the grouping of therestriction maps in the appended FIGS. 1 to 9 conform to the groupingsgiven in the column headed Composition in table 5. This is equally thereason why some of the probes are reproduced several times in thedifferent figures of the appended drawings.

Each of these mixtures may again be defined as including at least one ofthe new probes according to the invention. In other words, thediagnostic compositions according to the invention may be defined ascontaining:

-   1) at least the DNA of HPV-2d,-   2) at least the DNA of one of the HPVs 10b, 28 and/or 29,-   3) at least the DNA of one of the HPVs 17 and/or 24,-   4) at least the DNA of one of the HPVs 14, 15, 17, 19, 20, 21, 22    and/or 23,-   5) at least the DNA of one of the HPVs 15 and/or 17,-   6) the DNA of HPV-24,-   7) the DNA of HPVs 14 and 32,-   8) the DNA of HPV-31,-   9) the DNA of HPV-32,    it being understood that the DNAs of the nine groups are chosen in    such a manner as to in all circumstances be different from each    other.

In view of the great diversity of HPVs susceptible of being isolatedfrom the different forms of warts or other skin or mucous membranelesions, it is nevertheless preferable, for the diagnosis of each typeof disease mentioned in the table, to use mixtures containing more thanone or two HPV-DNAs, since it has been recognized that other HPV-DNAsmay equally intervene in the same type of affection. The diagnosis ofthe nature of the infection and its possible evolution will be that muchmore effective as the number of probes used is increased. In addition,hybridization assays carried out with the different mixtures of probeswill allow differential diagnoses with an equivalently greater degree ofprobability, of the disease from which the patient suffers.

In Table 5, only probes formed from HPV-DNAs isolated in thelaboratories of the inventors, have been mentioned. It goes withoutsaying that, because of the preceding, the different mixtures may beimproved by the addition of other HPV-DNAs obtained through the works ofother laboratories, once such are found from the various afflictions ofthe same kind presented by patients. For example, mixture 7 can only beimproved by the addition of any other HPV-DNAs encountered inepidermodysplasias verruciformis with the risk of transformation intointra-epithelial neoplasms and skin cancers. Note that in table 5,certain of the mixtures are presented as b characteristic to thediagnosis of the same diseases. It should nevertheless be recognizedthat the different mixtures make a distinction between those infectionsimplying little risk of cancerization, and those bearing a high risk ofit. For example, the hybridization of a viral preparation derived from apatient submitted for diagnosis, with mixture 7, suggests a greater riskof evolution to skin cancer than if the sample had hybridized to agreater degree with the probes of mixture 3.

Likewise, the cases of EV detected by mixture 5 implies a greater riskof cancerization than those cases detected with mixture 6. Mixture 4will discern EV cases having an even higher risk than those detected bymixture 5.

In the following is described other mixtures or cocktails of differentHPV-DNAs (or probes containing these HPV-DNAs or sequences of them),susceptible of being used in combination for the realization of globaldiagnoses of the different forms of papillomavirus infections, perhapsfor the purposes of determining a prognosis of the possible evolution ofthe affliction.

The preferred mixtures conforming to the invention are identified in theaforesaid table 5.

The aforesaid table equally indicates the natures of the affectionssusceptible of being more particularly diagnosed by the use of themixtures figuring on the left side of the table. It is to be recalledthat the restriction maps of other HPV-DNAs identified in the precedingtable are contained in the FIGS. 1 through 9.

It should be noted that HPV-IP2 may be considered as particularlyrepresentative of the probes useable for the detection of the risk ofdeveloping genital neoplasms and, in particular, cervical cancers.

The invention therefore concerns more particularly yet the “kits” fordiagnosis including at least 10 groups figuring in the groups numbered 1to 10 in the table under the heading “Designation of the mixtures”.

In the preceding, what was primarily under consideration was the use, asprobes, of whole, cloned HPV-DNAs. These, however, could be replaced bycloned fragments of these different DNAs, notably by the genes E1 or L1and by the genes E6-E7.

The basic principal of in vitro detections of HPV-DNAs will naturallyinvolve hybridizations operated under strict or less strict conditions.A procedural example follows, it being of course understood that thediagnostic assays described should in no way be considered as limitingthe conditions of use of the probes or mixtures of the probes accordingto the invention.

The object of examinations involving probes prepared from mixtures ofDNAs from cloned HPVs is to reveal an HPV and identify the type of HPVin a biopsy, in cells obtained by scraping a lesion, or in biopsysections fixed with Carnoy's mixture (ethanol, chloroform, acetic acid6:3:1) and included in paraffin. The examination requires the priorextraction of the DNA from the samples according to methods theprincipal of which is known and involves the analysis of this DNA bymolecular hybridization under strict or less strict conditions with theaid of radioactive probes (labeled with ³²P or ³⁵S) prepared frommixtures of HPV-DNAs. In general, each examination requires the use ofseveral mixtures of probes.

Several hybridization methods may be used. For example, the spothybridization method includes, after denaturation of the DNA, thedeposition of an aliquot of the DNA onto film supports (nitrocelluloseor Genescreenplus), the hybridization of each film under the usualconditions with a mixture of probes, and the detection of theradioactive hybrids by contact exposition of the hybridized film ontoradiographic film. Another possibility is replicatedculture-hybridization which involves agarose gel electrophoresisseparation of the DNA fragments resulting from treatment of the DNA byrestriction enzymes, the transfer of the fragments after alkalinedenaturation onto films (nitrocellulose or Genescreenplus) and theirhybridization under usual conditions with different mixtures of probes.The formation of radioactive hybrids is detected again by contactexposition of the hybridization support films onto radiographic film.

The radioactive probes are constituted of HPV-DNAs labeled by the “nicktranslation” method, or by RNAs prepared by transcription of viral DNAsinserted into a vector, for example of the type SP6. The use ofradioactive probes offers the advantage of great sensitivity, but thisdoes not exclude the use of non-radioactive probes, for examplebiotinylated and able to be recognized by antibodies either themselveslabeled or themselves recognizable by antibodies bearing enzymatic,fluorescent or other labeling.

The choice of probes depends on the nature of the patient sample. Thus,for example, in the case of a patient suspected of having EV, mixtures1, 2, 3, 4, 5, 6 and 7 would be used. Mixtures 1 and 2 would permitdifferential diagnosis between EV and skin warts. Probe 3, whichincludes the most frequently detected member of each of the three groupsof HPVs associated with EV, and probe 7, containing the DNAs of the HPVsassociated with cancers of EV, would permit diagnosis of the majority ofEV cases and, in particular, the identification of patients infectedwith the types of HPVs most likely to develop into cancers. The use ofmixtures 4, 5 and 6 would distinguish which type or types of HPV areinfecting the same patient.

The invention thus again concerns the sets or “kits” containing a numberof the probes indicated above, notably:

-   -   either the representatives of each of the 19 types and sub-types        of HPV-DNAs indicated above;    -   or mixtures of probes, preferably the diverse groups or mixtures        of probes which have been defined above, these kits being        intended for diagnostic examinations in vitro by hybridization        between the viral preparations obtained from patients, and the        diverse groups of mixtures.

As it goes without saying and as it results already anyway from thepreceding, the invention is in no way limited to those of itsembodiments and applications specifically anticipated, but, on thecontrary, embraces all variants, notably the reference in the claims toa designation HPV-DNA followed by a specific number, and to whichcorresponds an HPV-DNA whose restriction map has been provided in thedrawings, is to be understood as signifying that these claims cover allHPV-DNAs which have in common with this particular HPV-DNA, the power tobe classed in the same type, according to the definition which was givenabove, and all the more to HPV-DNAs belonging to the same sub-type.

It is also to be noted, particularly with regard to the DNA derived fromHPV-32 which appears in the drawings, that it is not split by any of thefollowing enzymes: AvaI, BalI, BamHI, ClaI, EcoRI, HindIII, NdeI, NruI,PvuII, SacI, SalI, SmaI, TthIII, or XmaI.

Note, too, that the following recombinant DNAs were filed with theC.N.C.M. (National Collection of Cultures of Micro-organisms of theInstitut Pasteur in Paris) the 30th of Nov. 1984, under the numberslisted here:

-   -   pBR322/HPV2d No. I-379    -   pBR322/HPV10bA No. I-380    -   pBR322/HPV10bB No. I-381    -   pBR322/HPV14a No. I-382    -   pBR322/HPV14b No. I-383    -   pBR322/HPV15 No. I-384    -   pBR322/HPV17a No. I-385    -   pHPV5 HindIIIB/HPV17b No. I-386    -   pBR322/HPV19 No. I-387    -   pBR322/HPV20 No. I-388    -   pBR322/HPV21 No. I-389    -   pHPV5 HindIIIB/HPV22 No. I-390    -   pBR322/HPV23 No. I-391    -   pBR322/HPV24a No. I-392    -   pBR322/HPV24b No. I-393    -   pBR322/HPV28 No. I-394    -   pBR322/HPV29 No. I-395    -   pBR322/HPV31 No. I-396    -   pSP64/HPV32 No. I-397    -   pLI55/IP2 No. I-450    -   pSP65/IP4 No. I-449

The invention yet more particularly the products of the expression ofgenes E6 and E7 from the different papillomaviruses which have beenevoked in the preceding and which may be used as the active principal ofvaccines able to induce, when administered in effective doses, theresistance of the host to the development of neoplasms associated withpapillomaviruses.

The invention equally concerns serums susceptible to being obtained byimmunization of a mammal, serums which may be used for the preparationof belays administrable in effective doses to a patient, notablyparenterally, these serums then being able to provoke a remission of theinfections induced by the corresponding types or sub-types ofpapillomaviruses.

It is pointless to emphasize the capacity of the specialist to obtainthe products of the polypeptidic expression of the kind in question,notably by the techniques of genetic engineering consisting of theincorporation of the E6 and/or E7 sequences into a vector under thecontrol of an appropriate promoter, then the transformation of acellular host, of which the papillomaviruses are likely to recognize thepromoters in question and to express the sequences associated with it.

The invention thus also concerns the compositions for pharmaceuticalusage containing the principles of the kind in question (expressionproducts or corresponding antibodies), in association with aphysiologically acceptable pharmaceutical vehicle. In particular, thislatter is constituted of an injectable saturated solution, in the casewhere the compositions of the kind in question will be administeredparenterally.

Lastly, reference is made to the articles whose bibliographicalreferences follow, which fill the requirement to describe the priorstate of the art to the extent that that proves useful to the completeunderstanding of this text by the reader. For this reason, the contentsof these articles should be considered as being part of the description.

BIBLIOGRAPHY

-   (1) Dürst, M. et al., 1983, Proc. Natl. Acad. Sci. U.S.A.,    80:3812-3815.-   (2) Coggin, J. R., Jr. et al., 1979, Cancer Res., 39:545-546.-   (3) Gissmann, L. et al., 1982, J. Virol. 44:393-400.-   (4) Green, M. et al., 1982, Proc. Natl. Acad. Sci. U.S.A.    79:4437-4441.-   (5) Heilman, C. A. et al., 1980, Virol. 36:395-407.-   (6) Jablonska, S. et al., 1972, Cancer Res., 32:583-589.-   (7) Jablonska, S. et al., 1982, Springer Semin. Immunopathol.    5:33-62.-   (8) Kremsdorf, D. et al., 1982, J. Virol. 43:436-447.-   (9) Kremsdorf, D. et al., 1983, J. Virol. 48:340-351.-   (10) Lutzner, M. A. et al., 1978, Bull. Cancer, 65:169-182.-   (11) Lutzner, M. A. et al., 1983, Lancet ii:422-424.-   (12) Migozzi, M. et al., 1965, Bull. Soc. Franc. Derm. Syph.    72:747-748.-   (13) Orth, G. et al., 1980, Cold Spring Harbor Conf. Cell    Proliferation, 7:259-282.-   (14) Orth, G. et al., 1981, J. Invest. Dermatol. 76:97-102.-   (15) Orth, G. et al., 1979, Cancer Res. 39:1074-1082.-   (16) Ostrow, R. S. et al, 1982, Proc. Natl. Acad. Sci. U.S.A.,    79:1634-1638.-   (17) Ostrow, R. S. et al., 1983, Ann. Acad. Dermatol. 8:398-404.-   (18) Pfister, H. et al., 1983, Cancer Res. 43:1436-1441.-   (19) Pfister, H. et al., 1983, J. Virol. 47:363-366.-   (20) Pfister, H. et al., 1981, Int. J. Cancer, 27:645-650.-   (21) Rueda, L. A. et al., 1976, Med. Cut. I.L.A. 2:113-136.-   (22) Ruiter, M. et al. J. Invest. Dermatol., 47:247-252.-   (23) Sutcliffe, J. G., 1978, Nucleic Acids Res. 5:2721-2728.-   (24) Tsumori, T. et al., 1983, J. Gen. Virol. 64:967-969.

1. A pharmaceutical composition for inducing remission of humanpapillomavirus (HPV) infection, comprising a. antibodies produced byimmunizing a mammal with an immunogenic composition wherein saidimmunogenic composition comprises a polypeptide encoded by a DNA of ahuman papillomavirus, wherein said DNA is about 7,000 to about 8,000base pairs, wherein said human papillomavirus is HPV-IP4, and whereinsaid polypeptide is encoded by a human papillomavirus gene selected fromthe group consisting of E1, E6, E7, L1 and L2; and b. a physiologicallyacceptable pharmaceutical vehicle.